1. Field of the Invention
The invention relates to neuron generation, regeneration and protection, more particularly to neurite outgrowth.
2. Description of Related Art
The invention provides means to induce and/or stimulate neuritogenic effects, which are useful in the medical field, more particularly in the treatment and/or palliation and/or prevention of disorders of the nervous system. More particularly, the invention provides means for inducing and/or stimulating neurite outgrowth, which are useful for inducing neuron differentiation, for example for the treatment of a neoplasm of the nervous system, as well as in regenerating impaired neurons, for example for the treatment of a neurodegenerative disease, disorder or condition or in the treatment of a microbial infection, or in protecting neurons from neurotoxic agents or oxidative stress.
The means of the invention are based on certain non-apoptotic rabies virus G proteins, more particularly on the cytoplasmic tail thereof.
During development of the nervous system, neurons extend axons over considerable distances in order to innervate their targets in an appropriate manner. This involves the stimulation in the cells of specific signaling pathways which can stimulate the activity of the growth cone.
While the developing nervous system, more particularly the developing central nervous system, is highly plastic, the adult nervous system, more particularly the adult brain, has more limited repair potential. Therefore, neurite-axon outgrowth and protection against degeneration are important factors to be considered to improve the outcome of a neurodegenerative disease, disorder or condition, such as an acute injury of the nervous system or a chronic neurodegenerative disorder. Products, which would be capable of inducing neurite outgrowth from such neuronal cells, would bring a very useful therapeutic and/or preventive and/or palliative solution to such diseases, disorders or conditions.
At the other side of the neuron developmental process, the proliferation of neuronal progenitors, which do not differentiate into matured neuronal structures, leads to nervous system neoplasm. Products, which would be capable of inducing neurite outgrowth from such progenic cells, would bring a therapeutic and/or preventive and/or palliative solution to such neoplasms.
The general idea on the infection of neuronal cells by a neurotropic virus is that it has no positive impact on neuron morphology, more particularly on neurite outgrowth.
Indeed, there are numerous examples showing that neurotropic viruses cause neuronal cell death by apoptosis. This concerns both DNA viruses, such as herpes viruses, and RNA viruses, either enveloped such as alphaviruses, bunyaviruses and paramyxoviruses, or unenveloped such as picornaviruses and reoviruses.
Rabies virus, more particularly attenuated rabies virus strains, has also been described as inducing neuronal apoptosis.
For example, WO 03/048198 relates to rabies virus G proteins and fragments thereof of at least 100 amino acids, which induce the disruption of the neuronal cell integrity and the formation of apoptotic bodies. These apoptotic bodies are capable of stimulating a humoral immune response, preferably a B-dependent humoral immune response. WO 03/048198 shows that:                attenuated rabies virus strain (such as the attenuated ERA strain) induces the apoptotic rupture of the cells it infects,        the apoptotic bodies thereby produced stimulate a humoral immune response, more particularly a B-dependent humoral immune response;        induction of apoptosis by a rabies virus strain is determined by the nature of its G-protein;        a rabies virus containing the G protein from an attenuated rabies virus strain (such as the attenuated ERA strain) is able to trigger apoptosis of human cells, whereas expression of the G protein from a pathogenic rabies virus (such as a Challenge Virus Standard -CVS- strain) is not (cf. more particularly example 5 and FIGS. 19 and 20 of WO 03/048198).Please see also Lay et al. 2003 and Préhaud et al. 2003.        
Thus, the G proteins of apoptotic rabies virus strains, such as the G protein of the attenuated ERA strain, are known to be useful in stimulating a humoral response, more particularly a B-dependent humoral immune response.
Since these particular G proteins induce the apoptosis of the cells they infect, they have also been proposed as candidate agents to eliminate undesirable cells by apoptotic rupture of the target cells.
It has also been described that the pathogenicity of a rabies virus strain is inversely correlated with its ability to induce apoptosis (cf. WO 03/048198; Ugolini 1995; Sarmento et al. 2005; Ugolini 2008; Jackson et al. 2008).
Therefore, the more virulent a rabies virus strain is, the less apoptotic.
The findings that virulent rabies virus strains, such as CVS strains, do not induce neuron apoptosis and thereby escape humoral detection explain why virulent rabies virus strains can propagate so extensively within the CNS before the appearance of signs and symptoms of the disease.
Further studies have been conducted to analyze the changes in gene expression pattern that are induced upon infection by a neurotropic virus, such as a rabies virus or herpes simplex type 1 (HSV-1).
These studies have brought the demonstration that post-mitotic human neurons, in the absence of glia, have the intrinsic machinery to sense virus infection, and that neurotropic viruses, such pathogenic rabies virus or HSV-1, induce the release of cytokines from post-mitotic human neurons (Préhaud et al. 2005). This cytokine release is believed to further contribute to the escape of neurons from apoptosis and in the consequent spreading of such neurotropic viruses.
Therefore, neuronal cell death mechanisms, as well as the capacity of neurons to raise an immune response upon viral infection, have been thoroughly investigated.
However, less is known about the processes involved in neurogeneration, neuroregeneration and neuroprotection, more particularly in neurite outgrowth from pre-mitotic neurons, such as neuronal progenitors or neoplastic neurons, or from degenerative neurons.
If one focuses on rabies virus, a schematic summary of the current knowledge would be that attenuated rabies virus strains are known to have medical applications due to the apoptosis they induce, and that virulent rabies virus strains are known to not induce apoptosis, but, to the contrary, to preserve the neuronal network, which favors their spreading.
While virulent rabies virus strains have been described as preserving the integrity of the neuronal network, they have also been reported as having a negative impact, or at the very least no positive impact, on neuronal morphology, more particularly on neurite outgrowth.
For example, the publication Guigoni and Coulon 2002 describes that the virulent rabies virus strain CVS-Gif-sur-Yvette do not induce neurite outgrowth from rat motoneurons (cf. for example, FIGS. 5A and 5B of this publication).
Negative impact on neurite outgrowth has also been reported. For example, the publication Scott et al., 2008 has reported that the pathogenic CVS-11 rabies virus strain induces beading of the dendrites and axons, i.e., the formation of vacuoles that are characteristic of a negative stress impact.
The invention provides means for the generation, regeneration and protection of neurons, which derive from certain pathogenic rabies virus strains, and which show surprising and unexpected properties.